Such assays are normally prepared and measured in sample plates or formats, including 96-well microtitre plates, petri dishes, gel matrices, membranes, glass slides and capillaries. The trend is towards higher throughput detection of samples and the use of smaller volumes in each of the samples, resulting in so- called miniaturised sample formats.
This requires the corresponding development of detectors capable of handling such miniaturised formats. This is particularly so in the case of high throughput screening (HTS) of biological assays as applied to drug discovery and the screening of drug candidates.
Such miniaturisation is achieved by arrarging samples for assay and detection in well plates in which typically there can be 96, 384, 864, 1536 or 3456 wells per plate, and sample volumes can vary from 200 microlitres to as little as 1 microlitre.
Alternative formats for the miniaturisation of assays include capillaries, microchannels or microfluidic structures, including microwells, which can be moulded or etched in substrates such as glass (eg silica or quartz) or plastic. In these alternative formats, the sample volumes can be of the order of nanolitres and picolitres.
In order to achieve high throughput screening it is necessary to interrogate large numbers of such samples simultaneously. In the case of fluorescence based assays, detection or interrogation consists of illuminating each sample with excitation light, and subsequently detecting the emitted fluorescence from each sample separately. Examples of fluorescent based processes include prompt fluorescence and time-resolved fluorescence where there is a time delay between photoactivation of the sample and emission, in the range, for example of ps to ms or more. A further process is fluorescence or luminescence energy transfer. In this process a molecule is activated, for example by excitation light, and transfers energy via eg resonance energy transfer or chemical transfer to a second molecule, which in turn emits light. This process can involve different or multiple secondary molecules, which can emit radiation over a range of wavelengths. Further examples of luminescent processes include phosphorescence, and chemi- and bio-luminescence. Wavelength ranges for all these processes include UV, visible, red and infra-red (approx 250–1200 nm).
In a typical arrangement, a scanning head with 96-channels simultaneously interrogates the 96-sites arranged in the 8×12 pattern of a 96-well microtitre plate. By stepping a higher well-density relative to a 96-channel scanning head, the remaining sites can be read. Eg 2×2 steps will cover 384-samples, 6×6 steps will cover 3456 samples, and so on. This allows the head to address higher density sample presentation formats.
Fluid samples, eg liquids or gels, can be placed in small sample sites, such as micro-capillaries or microchannels, which can be typically 100×100 um in section, and typically 1–100 mm long. The samples can be moved by pumps, or electrophoretically or electro-osmotically. Samples can be solid or a matrix, such as beads, agarose or microparticles, suspended or otherwise contained in a fluid medium, including a gel type format. Other samples can comprises suspensions or monolayers of cells.
Applications include cell biology, hybridisation techniques and immunoassays including binding assays. In such assays, materials can be labelled with a fluorescence marker for the purpose of identification. In a binding assay, a bound molecule can be separated from an unbound molecule, as between a solid and liquid medium. Further applications include electrophoresis or electro-osmosis fluids such as liquids, gels and media including agarose. Such applications can be run in miniaturised formats including micro-capillaries and micro- fluidic structures, where molecules or moities may be separated spatially by properties including molecular weight or charge and may also be labelled with fluorescent or luminescent tags or dyes for the purpose of detection and identification. The techniques described herein can be applied to the detection of biological compounds including proteins and nucleic acids, and in cell biology, processes such as cell signalling or cell binding can also be detected.
Separated molecules can be contained in a fixed matrix, such as a gel or agarose beads, or can be separated in a fluid such that the separated molecules or moities will flow at different rates through a medium and can be detected at a fixed point along the flow path as a series of emission peaks based on their time separation profile. It is important that emission detection methods posses high accuracy and sensitivity for the determination of such peaks, and the rapid and/or continuous and/or simultaneous measurement of samples.
A number of such assay techniques involve time changes in light emission. To measure such changes requires an ability to perform rapid, accurate, sensitive and repetitive readings, that is to perform kinetic measurements. In order to achieve high throughput it is necessary for a system to measure multiple samples simultaneously, requiring high resolution, high sensitivity and high signal detection efficiency.
Mostly the samples/assays are arranged to emit light in the middle range, although the quantities of light per sample can be very low.
A further issue with fluorescence-based assays is the problem of quench which causes a reduction in light emission. This can occur in samples and assays particularly those involving cells, due to chemical effects which interfere with the light signal, or due to coloured substances or particles in the sample or medium, which reduce the light signal. In certain applications, such as inhibition assays, a change or reduction in light signal is the feature of the assay requiring measurement.
FIG. 1(a) shows an array of 8×12 micro capillaries in a substrate, in which either a liquid, or molecules or moieties in a fluid, move in the X-direction. This is an example of an array of miniaturised samples such as will be referred to in this application.
FIGS. 1(b) and 1(c) show further examples of arrays that involve high density or miniaturised sample formats.
Imaging systems for detecting epi-fluorescence are known. Typically, as shown in FIGS. 1(a) to (c) of the accompanying drawings, an array of 96 fibre optic bundles is arranged to align with the centre of each of a plurality of sample sites on a sample plate—such as an 8×12 matrix 96 well plate, each well typically having a diameter of the order of 1 mm.